Summary
- Currently pollen tube data is analyzed by hand.
- Develop algorithms for automated analysis of pollen tube growth captured with GFP tagged videos.
- Verify current understanding of pollen tube dynamics spatially and temporally.
- Use four videos (600 frames) for quantifying the results.
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Introduction
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The current model of pollen tube growth. The enzyme, Rho GTPase, activates two downstream pathways: RIC4 and RIC3. RIC4 tagged with GFP relates polar tube growth to ROP1 activity. RIC3 regulates F-actin growth through the calcium gradient.
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Significance
- RIC4 over expression in N. tabacum results in different phenotype.
- Polar localization of RIC4 to plasma membrane (PM) results in polar tube growth; non-polar, directional tube growth.
Research Performed
- Analyze GFP tagged RIC4 video.
- Quantify displacement of PM and frequency of tip localization.
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Technical Approach
1. Forward Wavefront Region Growing
- Address non-polar localization that leads to non-polar growth.
- Relates a change in angle of the displacement vector to a non-polar localization.
- Overcomes non-rigid movement issues.
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1.1 Modified wavefront growth algorithm
- Propagate displacement vectors pixel by pixel outward from current points to next frame in video.
- Smooth vectors to provide better representation of displacement.
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Best match points from previous frame are mapped to new growth.
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A sample video of the algorithm. Displacement vectors (red arrows) are mapped to the points in the next frame (white border).
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2. Analysis of RIC4 Localization
- Verification of periodicity of RIC4 localization.
- Algorithm for Oscillation Analysis
- Histogram analysis to select threshold to report localization of RIC4.
- Use rotation to register all frames in identical direction.
- Generate signal for centroid movement corresponding to fluoresce.
- Compute Fourier transform to characterize frequency domain behavior.
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Experimental Results
Laser Microscopy Video
- Four videos of pollen tube growth with GFP-tagged RIC4.
- Three videos of 120 frames each, 10 s.
- One video of 240 frames, 20 s. with over expressed RIC4 stabilized with LatB.
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1. Wavefront Results
Frames displaying forward wavefront growing. From left to right: two frames of non-polar growth and one frame of polar growth. Note RIC4 localization to growth area.
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2. Signal Generation
Centroid movement accurately reports GFP fluorescence and RIC4 localization. (Lower Right) A signal generated from the sample frames.
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3. Oscillation
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3.1. Superimposed on Growth Rate
In correlation with previous research[2], tip growth lags behind RIC4 activity, and Rho GTPase activity. Arrows correspond to lagging peaks.
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3.2. Periodicity Analysis
Fourier analysis indicates common signal in all data. Signal is low frequency, and is not due to noise.
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Conclusions
- RIC4 localization directs both polar and non-polar pollen tube growth.
- Localization oscillation exhibits periodic behavior.
- Experimental results show potential of computer vision with fluorescently tagged videos.
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